<Production Techniques of Recombinant Proteins>
It has become possible to produce a variety of useful proteins including the ones occurring in an extremely small amount in an organism or having too low stability to be purified by the evolution of the recombinant expression techniques of a gene. The use of such techniques has realized not only the practical use of many recombinant proteins as pharmaceuticals or industrial enzymes, but also the elucidation of the stereostructures of proteins or the analysis of the interactions between proteins, and thus the comprehension of life has steeply proceeded. Furthermore, it has been possible to obtain useful genes by the steep proceeding of the genome studies on many biological species including human and the development of bioinformatics and PCR technologies. Therefore, unlike the conventional situations in which the cloning of genes has often been the rate determining factor of the studies, a great deal of time has now become required for developing the stable expression of a cloned gene, that is to say, the process for producing a protein encoded by the gene and in a large amount.
The hosts used for the production of recombinant proteins include Escherichia coli, yeast, cells derived from insects and mammals, but universal hosts for producing proteins which satisfy all of the needs have not yet been developed. Thus, trial and error must be carried out even now for the construction of production systems at every intended protein. By way of example, although Escherichia coli is the expression system used most popularly, it is at issue in the production of proteins having activities that the proteins produced often form insoluble inclusion bodies and post-translational modification such as glycosylation scarcely occurs distinct from eukaryotes. In addition, expression systems, in which eukaryotes such as yeasts or fungi are used as a host, are also used, but these systems are not always effective on all of the proteins and often difficult to conduct the expression of the activities or the complex post-translational modification of animal-derived proteins having complex structures. Furthermore, there has recently been often used an expression system with baculovirus and insect cells as a host cell. This system has many advantages in that the protein produced has been subjected to post-translational modifications such as phosphorylation and glycosylation and thus can be expressed with maintaining the original physiological activities, but the sugar chain structure of the secreted protein is different from that of the cells derived from mammals, so that problems such as the antigenicity of the recombinant proteins will be caused in pharmaceutical applications.
On the other hand, it is better to select an expression system with an organism related to the one from which a target protein is derived in order to produce the protein in the same state as in vivo, that is to say, in the same state in relation to the stereostructure of the protein maintained in an organism and the post-translational modifications such as phosphorylation, glycosylation and truncation. Therefore, an expression system having the cells derived from mammals as a host has been predominantly used in the expression of a protein which is derived from animals, in case that the post-translational modifications such as glycosylation to maintain the activity of the protein are required, the protein has a complicated structure, or the functions of the protein have not been identified. The expression system with animal cells is advantageous in that the protein can be subjected to precise post-translational modification, and thus proper folding can be expected for exerting its activities. Therefore, this is predicated the most appropriate system for the purpose of the biochemical analysis and functional analysis of proteins derived from animals.
Expression is classified into two types of the transient expression method in which a gene is expressed transiently and the stable expression method in which cells constantly expressing a gene are made. In the transient expression method, the transferred gene is transcribed and translated in the cells, and the expression of a protein is observed after several hours from the transfer and reaches its peak after two or three days. A method for amplifying the number of copies of a plasmid containing ori of SV40 by transferring the plasmid into cells such as COS cells in which the SV40 large T antigen gene is expressed is employed in order to increase the production amount of the protein, but it requires the transfer of the plasmid into the cells every time and thus the amount of the protein produced by this method is limited. On the other hand, when it is necessary to conduct the analysis with the cells in which the target protein has been constantly expressed or to produce the target protein in a certain amount, the stable expression method in which the transferred gene is inserted into the chromosome of the cells is selected. If a recombinant cell line having a high productivity of protein of interest has been established at all, all of the cells has expressed the target gene, so that a variety of analysis can be performed and culture in a large scale can also be conducted in order to produce a homogeneous recombinant protein. However, since the amount of gene expression largely varies among the recombinant cell lines due to the copies of the gene inserted into the chromosome of the cell or the position of the chromosome into which the gene has been inserted, a cell line having a high expression amount which can be used for the analysis or production must be selected. Most of the transformed cell clones, however, have extremely low expression amounts, and thus the selection of the high expression clone requires time-consuming and laborious operations.
As described above, in any cases of the production of a protein in either of the expression forms, various ideas have been managed to solve the problem that the production amounts of proteins in animal cells remain generally in lower levels as compared with those in the other recombinant expression host systems.
<Increase of the Expression Level of a Gene>
The expression level of an eukaryotic gene transferred into animal cells are regulated by various factors such as a DNA sequence which acts cis on the expression of the gene or the transcriptional regulatory factor which acts trans on the DNA sequence, the copy number of the gene, the insertion site of the transferred gene in chromosome, and the stability of mRNA (Dillon and Grosveld, Trends Genet., 9:134, 1993). The regulatory system has hitherto been analyzed from many aspects, and plasmid vectors for obtaining cell strains in which a gene is highly expressed have been developed on the basis of these results (Makrides S. C.; 1999). Typical information is now described below.
The cis-acting factors participating in the regulation of gene expression are DNA sequences, of which the typical ones includes promoter sequence and enhancer sequence. It has been examined thoroughly that a variety of transcriptional regulatory proteins which regulate transcription act as trans-acting factors on either sequence. The promoter sequence is adjacent to the upstream of the gene, and contains an essential region to basic transcription. The enhancer sequence may be present in a place apart from the gene or in an intron, and the orientation of the sequence is not fixed. Also, the enhancer sequence may often regulate the expression of a tissue-specific gene. The activities of promoters and enhancers can be generally detected for example by the transient gene transfer experiment. In order to highly express a foreign gene, it is important to arrange and utilize a potent promoter and an enhancer sequence effectively. The potent promoter sequence is often in close vicinity to the enhancer sequence, and includes for example SV40 early promoter, adenovirus major late promoter, mouse metallothionein I promoter, Raus sarcoma virus long terminal repeat and human cytomegalovirus (CMV) promoter.
In addition to the promoters and the enhancers, there are also cis-functional regulatory sequences for gene expression. These sequences are referred to as locus control region (LCR; Grosveld F., Cell 51:975, 1987), matrix attachment region (MAR; Phi-Van, Mol Cell Biol 10:2302, 1980), scaffold attachment region (SAR; Gasser, Trends Genet 3:16, 1987), or insulator element (Kellum, Cell 64:941, 1991), and are believed to act on the chromatin structure of chromosome. These regions have a function similar to the enhancer in view of point that these regions may function even if apart from the gene, but are distinguished from the enhancer sequences in that these regions can be detected only by the experiment of stably transferring a foreign gene into chromosome. Among these sequences, LCR is distinct from the enhancers in that it has functions depending on the site and orientation to the gene. Furthermore, the sequences called A box or T box and the topoisomerase II recognition sequence which are characteristically present in LCR and SAR are specific sequences which have not been found in the enhancer sequences or the promoter sequences (Klehr D., Biochemistry 30:1264, 1991).
HIRPE (Hot spot of Increased Recombinant Protein Expression) is a characteristic 5 kb DNA fragment which contains a sequence similar to MAR and an AT-enriched sequence, and is cloned from CHO cells in which a foreign gene is highly expressed (Koduri, K., Thammana, P., Patent No. WO 00/17337). It has been shown that the transformation of the CHO cells with an expression plasmid containing the DNA fragment linked with a foreign gene results in insertion of the plasmid into the particular site of chromosome and the increase of the expression level to several times. In addition, the expression augmenting sequence element (EASE; Morris, A. E., Patent No. WO97/25420) is also a factor discovered in CHO cells and is believed to have an effect of increasing the expression level of a foreign gene stably inserted into chromosome to several times. The activity is observed in a 14.5 kb DNA fragment cloned from the cells in which a foreign gene is highly expressed. No ORF which encodes regulatory factors is contained in the DNA fragment, and thus the effect of increasing the expression amount of the gene is thought due to the action of EASE, after having been inserted stably into chromosome, on a promoter or a enhancer sequence. Either method is a gene expression augmentation method with a certain specific DNA fragment, and thus these methods cannot be applied to the expression of the other foreign genes unless these DNA fragments are used.
Increased expression of a foreign gene can also be established by increasing the copy number of the gene in host cells. One of the methods for increasing the copy number of a foreign gene in transformed cells comprises co-transfecting the cells with a plasmid containing a selective marker gene and a large excessive amount of plasmid containing a foreign gene and no selective marker gene. According to this method, it is possible to obtain stably transfected cells in which a number of the foreign genes have been inserted into the chromosome (Kaufman, Meth. Enzymol., 185:537, 1990). However, since almost of the clones obtained by the transfection according to this method have only a few copies of the foreign gene, clones in which many copies of the foreign gene have been inserted must be screened, and thus time-consuming and laborious operations are required.
Another method for increasing the copy number of a foreign gene comprises gene amplification in the stably transfected cells which have been once selected. It is believed that the gene amplification naturally occurs in animal cells notwithstanding in a low frequency (Schimke R T, J Biol Chem, 1988, 263, 5989–92). There has been extensively employed a technique in which the foreign gene has preliminarily been transfected together with a gene which can be amplified into the host cells by taking advantage of the fact that the gene amplification is induced by exposing the cells to an appropriate selection pressure, the concentration of a selection agent is continuously increased, and thus the foreign gene is amplified together with the marker gene.
The marker gene generally used in this operation is a dhfr gene which encodes an enzyme dihydrofolate reductase, and the host cell which can be used is the CHO cell defective in dhfr activity. The gene is amplified by gradually increasing the concentration of the dhfr inhibitor methotrexate (MTX), and the target foreign gene in the vicinity thereof is also expected for amplification at the same time (Mammalian Cell Biotechnology, Ed. Butler, M. IRL Press, P 79). Furthermore, it has been described that the foreign gene can be amplified to the number of copies of 2000 by increasing the MTX concentration in three steps (Bebbington, C. and Hentschel, C., Trends Biotechnol., 3., 314 1985). However, this method has the problems that it is time-consuming and can be applied only to dhfr gene deficient cells. In addition, the agent for selection is expensive, and thus it is not preferred to add the agent in the large-scale culture of recombinant animal cells. Furthermore, it has been also indicated that the gene once amplified by this method is unstable and tends to be deleted under the condition of the non-addition of the agent (“Gene”, Ver. 6, Translated by Tsuguhiko Kikuchi, P 845–848, Tokyo Kagaku Dojin).
<Establishment of Stable Cell Lines in Which Genes are Highly Expressed>
A selective marker gene is required for selecting the animal cells into which a foreign gene has been stably transferred. Various kinds of marker genes are used for selection and are classified largely into two groups. One of the groups includes genes of, for example, hypoxanthine-guanine phosphoribosyl transferase (HGPRT), thymidine kinase (TK), dihydrofolate reductase (DHFR), and adenine phosphoribosyl transferase (APRT), and only the cells deficient in the enzymatic activity corresponding to each of the genes may be used as the host. Since auxotrophy in such cells deficient in these enzymatic activities will be recovered by transferring the corresponding genes, it is possible to select the cell strains into which a foreign gene has been transferred. Another group is a gene group for conferring resistance to antibiotics and drugs which inhibit the growth of the cells. Specifically, it includes, for example, mutated DHFR for conferring resistance to methotrexate, xanthine-guanine phosphoribosyl transferase (gpt) for giving resistance to xanthine, and the transposon Tn5-derived aminoglycoside 3′-phosphotransferanse (neo) for giving resistance to drugs such as geneticin (G418), gentamycin, kanamycin, and neomycin. There have recently been developed also the genes for affording resistance to zeocin or hygromycin. These genes may be used as selective marker genes for all of the animal cells.
Cycloheximide (CYH) is a protein synthesis inhibitor, and an application example with the CYH resistant gene as a selective marker includes an example with yeast. It has been elucidated in yeasts that CYH acts on the L41 protein, the sub-unit of ribosomal protein, to inhibit the biosynthesis of the protein and become sensitive to CYH when the amino acid at the position 56 of the protein is proline while non-sensitive to CYH when the amino acid is glutamine (Kawai S. 1992, J. Bacteriol., 174, 254–262). Based on this information, it has been demonstrated that when the gene of the L41 protein is cloned from CYH sensitive yeasts such as Candida utilis and Phaffia rhodozyma and mutation by substitution is transferred to construct a gene of the L41-Q type, which is then transferred into the original yeasts, these yeasts become resistant to CYH (Kondo K., 1995, J. Bacteriol., 177, 24, 7171–7177; Kim I.-G., 1998, Appl. Environ. Microbiol., 64, 5, 1947–1949). However, there have been described no applications to higher eucaryotes such as animal cells.
Foreign gene transferring vectors into animal cells generally possess either one or both of these marker genes, and vectors into which a green fluorescent protein (GFP) has been contained are also used in order to select a high expressing strain more easily.
While clones in which the desired protein is highly expressed must be selected from many transfected cells obtained with the above described selective marker genes, a foreign gene in most of the transfected cells is generally expressed in an extremely low level and is screened with an extreme difficulty.
Therefore, there have been described several methods of directly selecting high-expression transformed cells. One of the methods comprises co-transfecting cells with a foreign gene and a dhfr gene, directly culturing the cells in a culture medium containing Mtx in a high concentration, and thus selecting the transduced cells in which dhfr is expressed in a high level. Many of the cells selected by this method express a foreign gene in a high level (Page M J, Sydenham M A, Biotechnology 9, 1991, 64–68). However, high-expression cells obtained directly by single step using a culture medium containing a selecting agent in a high concentration are inferior in growth and stability and thus inappropriate to the use for the production of proteins for a long period of time. Furthermore, among the recombinant cells obtained by the direct selection with Mtx, there may be preferentially selected cells which contain dhfr enzyme of which the sensitivity to Mtx have been changed or are hardly affected by Mtx.
The second method comprising expressing a gene, which encodes the desired protein, together with a selective marker gene by using one promoter and thus obtaining efficiently high-expression cells has also been used. In this method, a vector which is constructed to express mRNA consisting of the foreign gene at the 5′-side and the selective marker gene at the 3′-side is used. It is generally expected that since the translational efficiency of the gene at the 3′-terminus of a polycistonic mRNA is bad, the polycistonic mRNA is expressed in a high level in transducing cells selected, and the desired protein is expressed in a high level (Kaufman R J, Murtha P, Davies M V EMBO J, 6:187 1987). However, when the translational efficiency of the selective marker at the 3′-side is largely affected depending on the genes at the 5′-side or the expression of the selective marker gene at the 3′-side remains in an extremely low level, risks may happen that the transformed clone is not selected or a cell having an increased translational efficiency of the selective marker gene due to the partial deletion of the gene at the 5′-side is selected. Internal ribosome entry site (IRES) is a sequence discovered in, e.g. virus derived RNA, and has been confirmed to promote the binding of ribosome to mRNA and the initiation of translation (Kaufman R. J., Nucleic Acid Res 19:4485, 1991). It is possible to enhance the translational efficiency of the gene at the 3′-side on the polycistonic mRNA by taking advantage of this property, and vectors in which the high-expression cells are selected relatively easily have been developed with IRES.
As the third method, there is also mentioned a method in which the expression of the selective marker gene is artificially lowered for expecting the increase of the expression of the gene transferred into the transfected cell at the same time. For instance, an intron is arranged at the 5′-side of a gene in which the desired protein is encoded, and the selective marker gene is arranged within the intron. Thus, only the mRNA with the complete length which is present in a low frequency and will not be spliced produces selective marker proteins. It has been shown that the transcript of a gene containing the selective marker gene in the intron is expressed in a high level in such transduced strains selected by the plasmids, and thus the desired protein is expressed in a high level (Clorley, Craig W., WO 96/04391). Furthermore, there has been also reported a method for expressing a foreign gene in a high level in a selected transducing strain by diminishing the expression of a selective marker gene by modifying the DNA sequence around the translation initiating codon of the selective marker gene and lowering the translational efficiency of the gene (Reff Mitchell E. WO 98/41645).